[CANCER RESEARCH 63, 7679 –7688, November 15, 2003]
Small Interfering RNA-Mediated Reduction in Heterogeneous Nuclear
Ribonucleoparticule A1/A2 Proteins Induces Apoptosis in Human
Cancer Cells but not in Normal Mortal Cell Lines
Caroline Patry, Louise Bouchard, Pascale Labrecque, Daniel Gendron, Bruno Lemieux, Johanne Toutant,
Elvy Lapointe, Raymund Wellinger, and Benoit Chabot
Département de Microbiologie et d’Infectiologie. Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada
ABSTRACT
To prevent their recognition as DNA breaks, the ends of linear chromosomes are organized into telomeres, which are made of proteins bound
to telomere-specific, double-stranded repeats and to single-stranded DNA
extensions, the G-tails. The mammalian heterogeneous nuclear ribonucleoparticule A1 and A2 proteins can bind with high affinity to such
G-tails. Moreover, previous work established that in certain mouse cells a
severe reduction in the level of A1 is associated with shortened telomeric
repeat tracts, and restoring A1 expression increases telomere length. Here,
we document that the expression of A1/A2 proteins is elevated in a variety
of human cancers, whereas A1/A2 expression is lower or absent in normal
tissues. To determine whether the status of A1/A2 proteins could be
improved from cancer markers to cancer targets, we used small interfering RNA-mediated RNA interference to elicit a reduction in A1/A2 proteins in a variety of human cell lines. We show that this treatment
provoked specific and rapid cell death by apoptosis in cell lines derived
from cervical, colon, breast, ovarian, and brain cancers. Cancer cell lines
that lack p53 or express a defective p53 protein were equally sensitive to
a small interfering RNA-mediated decrease in A1/A2 expression. The
reduction in A1/A2 levels in HeLa cells was associated with a change in the
distribution of the lengths of G-tails, an event not observed when apoptosis
was induced with staurosporine. Remarkably, comparable decreases in
the expression of A1/A2 in several mortal human fibroblastic and epithelial cell lines did not promote cell death. Thus, manipulating the level and
activity of A1/A2 proteins may constitute a potent and specific approach
in the treatment of human cancers of various origins.
INTRODUCTION
The telomeres are essential for protecting chromosome ends from
degradation, recombination, and fusion events. Telomeric DNA consists of a variable number of TAGGGT repeats in double-stranded
form, followed by a single-stranded overhang of the guanine-rich
strand, making up the 3⬘-end of the DNA (1). In human cells, the size
of this overhang is estimated at 150 –300 nucleotides, and at least a
portion of this extension is thought to invade the proximal doublestranded telomeric DNA to form a T-loop (2, 3). Telomeric repeat
DNA is bound by distinct protein complexes. For example, TRF1 and
TRF2 bind directly to double-stranded telomeric DNA, interact with
other proteins, and are important for telomere homeostasis (4, 5).
Proteins that interact specifically with the terminal single-stranded
repeats include the hnRNP1 A1 and A2 proteins (6 – 8), as well as the
hPot1 protein discovered recently (9, 10). Finally, the ribonucleoproReceived 6/27/03; revised 8/13/03; accepted 9/15/03.
Grant support: Telogene Inc.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Raymund Wellinger or Benoit Chabot, Université de Sherbrooke,
Faculté, de Médecine, Département de Microbiologie et d’Infectiologie, 3001, 12e avenue
Nord, Sherbrooke, Québec, Canada J1H 5N4. Phone: (819) 564-5321; Fax: (819) 5645295; E-mail: [email protected] or [email protected].
1
The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoparticule;
siRNA, small interfering RNA; RNAi, RNA interference; oligo, oligonucleotide; FBS,
fetal bovine serum; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end
labeling; PARP, poly(ADP-ribose) polymerase; T-OLA, telomere oligo ligation assay.
tein enzyme telomerase interacts with the very terminus of the singlestranded telomeric repeats and extends the G-rich strand, a process
that helps to solve the end-replication problem. However, although an
absence of telomerase can lead to a gradual loss of telomeric sequences and eventual cell division arrest, replicative senescence is
associated more with changes in the terminal capping structure itself
than with the overall length of telomeric repeat DNA (11). Consistent
with this view, a loss of the single-stranded G-rich extension has been
shown recently to correlate with senescence in human fibroblasts
(12, 13).
Current cancer models for human cells postulate that telomeric
decay imposed by active cell growth in the absence or low levels of
telomerase successively triggers p53-dependent cell-cycle arrest, escape from the arrest, and continued divisions that increase the frequency of dicentric chromosome formation (14, 15). The resulting
genetic instability may then promote mutagenic events until mutant
cells that are able to maintain stable telomeres emerge and develop
into a tumor. In ⬃85% of all of the tumors, stabilized telomeres are
thought to be a direct consequence of the reactivation of the telomerase enzyme (16). However, distinct mechanisms involving other
pathways (e.g., Alternative Lengthening of Telomeres or ALT) have
also been uncovered (17, 18). Therefore, the presence of telomeric
repeats for the capping function is absolutely essential for the growth
of cancer cells, irrespective of their origin.
Many studies aimed at reversing the neoplastic phenotype of cells
have targeted the activity of proteins involved in telomere biogenesis.
For example, the expression of a catalytically inactive form of telomerase in human cancer cell lines promotes telomere shortening, ultimately leading to growth arrest and cell death (19 –21). The use of
telomerase inhibitors to promote telomere shortening in cancer cells is
also being pursued (22–26). Proteins involved in the capping function
of telomeres represent interesting targets as well (27, 28), and would
include proteins that recognize the single-stranded G-rich telomeric
extensions. It is conceivable that approaches that will interfere with
the capping function of telomeres in cancer cells may lead to rapid cell
growth arrest and cell death. For example, the double-stranded DNA
binding telomeric protein TRF2 may play a role in this capping
function based on its role in T-loop formation and in the ability of a
dominant-negative version of TRF2 to promote chromosome fusions
and rapid p53-dependent apoptosis (5).
hnRNP proteins are among the most abundant nuclear proteins in
mammalian cells. There are ⬎20 hnRNP proteins, all of which can
associate with precursor mRNAs, and many influence pre-mRNA
processing and other aspects of mRNA metabolism and transport (29).
The best-characterized protein of the group is hnRNP A1 (hereafter
referred to as A1). A1 also binds with high affinity to telomeric
single-stranded DNA sequences (7, 30) and can interact simultaneously with telomerase RNA in vitro (31). Importantly, defective A1
expression in mouse erythroleukemic cells results in short telomeres,
which can be lengthened by restoring normal levels of A1 or by
expressing UP1, a smaller version of A1, which lacks the A1 splicing
function (7). Overexpressing A1 also elicits telomere elongation in
7679
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
human HeLa cells (7). A close homologue of A1, the hnRNP A2
protein (referred to as A2), shares 69% amino acid identity with A1.
Although A2 as well can bind specifically to single-stranded telomeric
sequence in vitro (8), its role in telomere biogenesis in vivo has not yet
been confirmed. For both A1 and A2, less abundant splice variants
have been described (A1B and B1, respectively). Interestingly, A1
expression is high in proliferating and transformed cells (32). A1 is
overexpressed in colon cancers (33) and in mononuclear cells from
leukemia patients (34), whereas the A2/B1 proteins have been used
as early markers for the detection of lung and pancreatic cancers
(35– 40).
The purpose of this study was to further examine the relationship
between A1/A2 expression and different types of human cancers.
Immunohistochemistry with A1/A2 antibodies revealed a strong correlation between A1/A2 expression and cancer cells, with little expression in normal cell types. In addition, we used RNAi (41) to elicit
a reduction in the level of A1/A2 proteins in human cell lines. The
results show that the combined reduction in A1 and A2 expression
promotes apoptosis in all of the cancer cell lines tested. In sharp
contrast, a similar decrease in A1/A2 protein levels in normal mortal
cell lines has no significant effect on cell growth. These results are
consistent with the view that the A1/A2 proteins are functional homologues playing a role as mammalian telomeric capping factors.
Therefore, abrogating A1/A2 expression would represent a powerful
and specific approach to prevent the growth of cancer cells.
MATERIALS AND METHODS
Anti-hnRNP Antibodies. Rabbit polyclonal sera raised against a peptide
unique to the hnRNP A1 protein (ASASSSQRGR, anti-A1) or against a
peptide common to both hnRNP A1 and A2 proteins (KEDTEEHHLRDYFE,
anti-A1/A2) were used for the immunohistochemical studies. Peptide synthesis
and antibody production was carried out initially by the Service de Séquence
de Peptide de l’Est du Québec, (CHUL, Ste-Foy, Québec, Canada). The
specificity of each serum was confirmed by ELISA and Western analyses
(Fig. 1).
Immunohistochemistry. The normal tissue screen was performed on 10
different normal human tissues using both sera. Two different sections of the
same tissue sample were treated independently with each serum. Three different samples per cancer type were screened using the anti-A1 and the antiA1/A2 sera. Immunohistochemistry was conducted by LifeSpan BioSciences
Inc. (Seattle, WA). Briefly, serial dilution studies demonstrated that on paraffin-embedded, formalin-fixed tissues, the highest signal-to-noise ratios for the
Fig. 1. hnRNP A1/A2 expression in cancer and normal tissues. A, Western analysis of
anti-A1 antibody was obtained at dilutions of 1:100 and 1:250, and for the
total HeLa protein extracts with rabbit polyclonal antibody raised against the A1 peptide
anti-A1/A2 antibody at dilutions of 1:1000 and 1:2000. These antibodies were or the peptide common to both A1 and A2 proteins. The position of A1, A2, and their
B
used as primary antibodies, and Vector antirabbit secondary antibody (BA- respective isoforms A1 and B1 is shown. Immunohistochemistry analysis of A1/A2
expression in lung tissue from (B) normal patient and (C) patient with lung adenocarci1000), Vector ABC-AP kit (AK-5000) with a Vector Red substrate kit (SK- noma. Immunohistochemistry analysis of A1/A2 expression in a pancreatic tissue from
5100) were used to produce a fuchsia-colored deposit. Tissues were also (D) normal patient and (E) patient with pancreatic adenocarcinoma. F and G represent
stained with a positive control antibody (CD31) to ensure that the tissue immunochemistry analysis with the anti-A1 antibody in breast benign duct and infiltrating
antigens were preserved and accessible for immunohistochemical analysis. The ductal carcinoma, respectively. Magnification, ⫻40.
negative control consisted of performing the entire immunohistochemistry
procedure on adjacent sections in the absence of primary antibody. Slides were
supplemented with 10% FBS. HIEC cells were grown in Opti-MEM I suppleimaged using a DVC 1310C digital camera coupled to a Nikon microscope.
Cell Cultures. HeLa S3, HCT 116, HT-1080, MCF-7, and CCD-18Co mented with 5% FBS. PA-1 and SK-OV-3 cells were grown in DMEM-F12
cells were obtained from the American Type Culture Collection. BJ foreskin supplemented with 10% FBS. MCF-7 cells were grown in ␣MEM supplenormal fibroblasts were kindly provided by James Smith (Baylor College of mented with 10% FBS, 0.1 mM nonessential amino acids, and 10 ␮g/ml bovine
Medicine, Houston, TX). HIEC cells were provided by Jean-François Beaulieu insulin. HT-1080 and CCD-18Co cells were grown in ␣-MEM supplemented
(Université de Sherbrooke, Québec, Canada). PA-1 and SK-OV-3 cells were with 10% FBS, Earle’s salt, 1 mM sodium pyruvate, and 0.1 mM nonessential
obtained from Claudine Rancourt (Université de Sherbrooke). U-373 MG and amino acids.
siRNAs. Oligos were purchased from Dharmacon Research, Inc. (LafayBJ-TIELF cells were a gift from David Fortin (Université de Sherbrooke) and
Sam Benchimol (Ontario Cancer Institute, Toronto, Ontario, Canada), respec- ette, CO). Sequences were selected from the targeted mRNA sequences and
tively. HCT 116 p53⫺/⫺ were a gift from Bert Vogelstein and Kenneth W. submitted to a BLAST search to ensure that only one human gene was targeted.
Kinzler (Johns Hopkins Medical Institute, Baltimore, MD). HeLa S3 and Seven siRNAs targeting the human A1 mRNA (GenBank accession no.
U-373 MG cells were grown in DMEM supplemented with 10% FBS. HCT NM_002136) were tested. They covered nucleotides 107–127 from the start
116 and HCT 116 p53⫺/⫺ cells were grown in McCoy’s 5A medium sup- codon (A1–1), 135–155 (A1–2), 154 –174 (A1–3), 217–237 (A1– 4), 404 – 424
plemented with 10% FBS. BJ and BJ-TIELF cells were grown in ␣-MEM (A1–5), 601– 621 (A1– 6), and 757–777 (A1–7). Nucleotides 117 and 118 in
7680
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
A1–1 were changed to CG to yield A1–1M. Five siRNAs were directed at the phenol-chloroform extraction. Samples were ethanol-precipitated and dishnRNP A2 mRNA (GenBank accession no. NM_002137) and were from solved in 6 ␮l of 10 mM Tris (pH 8)-1 mM EDTA buffer. Three ␮l of each
nucleotides 48 – 68 (A2–1), 57–77 (A2–2), 298 –318 (A2–3), 615– 635 (A2– 4), reaction was mixed with 4 ␮l of formamide dye, denatured by heating at 90°C,
and 922–942 (A2–5). Before transfection, siRNA duplexes were prepared by and quenched on ice before loading onto an 8% acrylamide-urea gel. Gels were
annealing the complementary oligos. The final concentration of the siRNA exposed, and ligation products were quantified using Quantity One software
duplex was 50 ␮M in 20 mM KCl, 6 mM HEPES-KOH (pH 7.5), and 0.2 mM (Bio-Rad). The value of the intensity of each band in the ladder of ligation
products was normalized for the number of concatenated oligo in the product.
MgCl2.
siRNA Transfections. The transfection procedure described below indi- The values obtained for ligation products containing 3 and 4 oligos were then
cated that typically at least 80% of the cells took up a fluorescent oligo. The grouped and compared with products containing 5 and more oligos by plotting
day before transfection, exponentially growing cells were trypsinized and their abundance relative to the total intensity of all of the selected products.
seeded into six-well plates. Transfection was performed on 30 –50% confluent This procedure allows for a clearer visualization of the loss of longer ligation
cells using Oligofectamine (Invitrogen) according to the manufacturer’s in- products.
structions and at the indicated total siRNA concentrations: HeLa S3 (80 nM),
HCT 116 (40 nM), HCT 116 p53⫺/⫺ (40 nM), HT-1080 (20 nM), PA-1 (10 RESULTS
nM), U-373 MG (10 nM), SK-OV-3 (40 nM), MCF-7 (80 nM), HIEC (80 nM),
BJ (80 nM), BJ-TIELF (80 nM), and CCD-18Co (80 nM). Briefly, the siRNAs
hnRNP A1/A2 Expression in Cancer and Normal Tissues. We
(in 10 ␮l) were mixed with 175 ␮l of OPTI-MEM-I (Invitrogen), whereas used rabbit polyclonal antibodies to investigate the expression of
Oligofectamine was mixed with OPTI-MEM-I (4 ␮l and 11 ␮l, respectively). hnRNP A1 and A2 in various human cancer biopsies and normal cell
The transfection reagent and the siRNAs were then mixed and incubated at
types. Immunohistochemistry was performed with the anti-A1 antiroom temperature for 20 min before being applied to cells. Fresh medium was
body, which recognizes the A1 and A1B proteins, and with the
added, and a second transfection at the same concentration of siRNAs was
B
conducted 24 h later. At least three independent experiments were carried out anti-A1/A2 antibody, which recognizes the A1/A1 /A2/B1 proteins
(Fig. 1A). For each cancer, three samples from different patients were
for each cell line, and typical results are shown.
Cell Growth and Viability Measurements. At the indicated times after used. High levels of nuclear A1/A2 expression were observed in all
the first transfection, both adherent and floating cells were harvested and three of the biopsies from breast, small cell lung, and ovarian carcicounted. Cell viability was evaluated by trypan blue dye exclusion. The
nomas (Table 1; Fig. 1). For lung adenocarcinomas, pancreas carcinumber of population doublings after transfection was calculated for each nomas, and skin melanomas, two biopsies scored as high, and one
sample using the equation: PD ⫽ log (Nf/N0)/log 2, where Nf is the number of sample scored as moderate. Two biopsies of prostate carcinomas were
cells at the end of the experiment and N0 equals the number of cells at the
strongly positive for nuclear A1/A2 expression, but one biopsy rebeginning of the experiment. TUNEL labeling was performed using the Apmained negative. Thus, most cancer tissues examined displayed relopTag kit (Intergen; S7110), according to the manufacturer’s instructions.
atively strong levels of nuclear A1/A2.
Propidium iodide (1 ␮g/ml) was used as a nuclear counterstain to visualize the
The expression pattern of nuclear A1/A2 in normal human tissues
whole cell population. Cells were visualized by fluorescence microscopy.
was
considerably different (Table 2; Fig. 1). Except for the basal layer
For DNA content analysis, both floating and adherent cells were recovered,
of skin tissue, which expressed high-level of A1/A2, most other
fixed in 80% cold ethanol, incubated at room temperature for 5 min, and stored
at ⫺20°C. The cells were washed with PBS A and treated with RNase A for normal tissues examined expressed low to undetectable levels of
30 min at 37°C (20 ␮g RNase A, 5 mM EDTA, and 0.5% BSA in 1 ml PBS A1/A2. Occasional A1/A2 expression was noted in some neurons, and
A). The cells were stained with propidium iodide (50 ␮g/ml) for 5 min at room significant expression was detected in kidney epithelia and endothetemperature and read on a Becton Dickinson FACScan using the CellQuest lium, in bile duct, in intestinal macrophages, in lymphocytes, and in
software. For each sample, at least 10,000 events were analyzed for DNA mesothelium of the spleen. The higher expression of the A1/A2
content.
proteins in the nucleus of tumor cells as compared with normal cells
Western Blotting. Whole cell extracts were prepared by lysing cells in
confirms A1/A2 as potentially broad markers for cancer detection.
Laemmli sample buffer [1⫻ ⫽ 10% glycerol, 5% ␤-mercaptoethanol, 2.3%
The binding A1/A2 to telomeric sequences and the implication of A1
SDS, 62.5 mM Tris-HCl (pH 6.8), and 0.1% bromphenol blue]. Equal amounts
of each sample (15–25 ␮g of total protein) was loaded onto a polyacrylamide
gel. Western blotting was performed according to standard protocols using the
Table 1 hnRNP A1/A2 expression in cancer tissues
following dilutions for primary antibodies: 1:5000 for the anti-A1/A2 antibodies, 1:500 for the anti-PARP antibodies (Biosource; AHF0262), 1:100 for the
A1/A2
active caspase-3 antibodies (Chemicon; AB3623), and 1:500 for the antiTumor
Sample
expression
procaspase-3 antibody (Biosource; AHZ0052).
Breast cancer
1
⫹⫹⫹
Telomere G-Tail Extension Analysis (T-OLA). The T-OLA assay was
2
⫹⫹⫹
3
⫹⫹⫹
carried out as described (42). For the staurosporine experiment, cells were
Colon carcinoma
1
⫹⫹⫹
treated for 28 h with staurosporine at a concentration of 1 ␮M at a final
2
⫹
concentration of 1.0% DMSO. Control cells were treated with DMSO 1.0%.
3
⫹⫹⫹
Briefly, genomic DNA was prepared by standard cell lysis protocols. Oligo
Lung adenocarcinoma
1
⫹/⫺
2
⫹⫹
(CCCTAA)3 was end-labeled and phosphorylated by T4 polynucleotide kinase
3
⫹⫹⫹
in the following reaction mixture: 0.16 ␮M of oligo, 1.6 ␮M of [␥-32P]ATP
Small cell lung carcinoma
1
⫹⫹
(3000 Ci/mmol, 10 mCi/ml), 70 mM Tris (pH 7.6), 10 mM MgCl2, 5 mM DTT,
2
⫹⫹
and 20 units of T4 polynucleotide kinase in a final volume of 50 ␮l. The
3
⫹⫹
Ovary carcinoma
1
⫹⫹
reaction was allowed to proceed for 40 min at 37°C. Then 1 ␮l of 0.1 M ATP
2
⫹⫹
and an additional 10 units of kinase were added before another 20-min
3
⫹⫹⫹
incubation period. The enzyme was then heat-inactivated at 65°C for 20 min.
Pancreas carcinoma
1
⫹/⫺
The oligo was precipitated with ethanol and dissolved in water. Hybridization
2
⫹⫹⫹
3
⫹⫹
was conducted in a 20 ␮l volume containing 10 ␮g of undenatured DNA, 0.5
Prostate carcinoma
1
⫹⫹
pmol of oligo, 20 mM Tris (pH 7.6), 25 mM potassium acetate, 10 mM
2
⫺
magnesium acetate, 10 mM DTT, 1 mM NAD, and 0.1% Triton X-100 at 50°C
3
⫹⫹
for 12 h. Forty units of Taq ligase (New England Biolabs) and 2 ␮l of fresh 10
Skin melanoma
1
⫹⫹
2
⫹⫹
mM NAD stock were added, and the ligation reaction was allowed for 5 h at
3
⫹/⫺
the same temperature. Reactions were ended by adding 30 ␮l of water and by
7681
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
Table 2 hnRNP A1/A2 expression in normal tissues
Tissue
Cell type
A1/A2
expression
Neurons (some)
⫹⫹
Neutrophils
⫺
Astrocytes, microglia, oligodendrocytes, endothelium,
⫺
vascular smooth muscle
Heart
Cardiac myocytes, endothelial cells, vascular smooth
⫺
muscle, fibroblasts
Kidney
Endothelium, thick and thin loop of Henle, collecting
⫹/⫹⫹
duct epithelium, glomerular capillary endothelium and,
vascular smooth muscle
Bowman’s capsule epithelium, podocytes, proximal and
⫹/⫹⫹
distal convoluted tubules
Mesanglial cells
⫺
Liver
Hepatocytes, endothelium, lymphocytes, vascular
⫹
smooth muscle
Bile duct
⫹⫹
Fibroblasts
⫺
Macrophages, Kupffer cells
⫺
Lungs
Pneumocytes, fibroblasts, endothelium, mesothelium
⫹
Alveolar macrophages
⫺
Pancreas
Endothelium, vascular smooth muscle, fibroblasts,
⫺
adipocytes
Peripheral islets cells
⫺
Acinar epithelium
⫹/⫺
Pancreatic duct
⫹/⫺
Skeletal muscle Myocytes
⫹
Vascular smooth muscle
⫺
Endothelium
⫹
Fibroblasts
⫺
Skin
Squamous epithelium (basal layer)
⫹⫹/⫹⫹⫹
Squamous epithelium (nucleated layer), superficial
⫹
dermal fibroblasts, endothelium, lymphocytes
Stratum lucidum, eccrine sweet glands
⫺
Subcutaneous glands
⫺
Vascular smooth muscle
⫺
Mast cells
⫺
Small intestine Neuroendocrine cells, epithelium (bases of crypts)
⫺
Villi columnar epithelium, lymphocytes
⫹
Goblet cells, Schwann cells
⫺
Macrophages
⫹⫹
Smooth muscle
⫹/⫺
Fibroblasts, ganglion cells, endothelium
⫹/⫺
Spleen
Smooth muscle, macrophages
⫺
Lymphocytes, mesothelium
⫹/⫹⫹
Fibroblasts
⫺
Neutrophils
⫹
Endothelial cells
⫺
Brain
in telomere biogenesis raises the possibility that A1/A2 proteins may
not only constitute useful cancer markers but may also play a crucial
role in the maintenance of the transformed state, possibly as telomeric
capping factors.
RNAi on hnRNP A1 and A2 in HeLa S3 Cells. If the A1/A2
proteins are involved in the formation of a telomeric cap, abrogating
their expression could induce telomere uncapping, which in turn may
promote cell growth arrest and rapid cell death (27). To test this
hypothesis, we used siRNAs to promote a specific reduction in the
level of A1 and/or A2 proteins in human cancer cells. Doublestranded siRNAs against A1 or A2 were individually introduced into
HeLa S3 cells by performing two successive applications of siRNA
(80 nM) at a 24-h interval. Seven different siRNAs targeting A1 and
5 siRNAs targeting A2 were tested. siRNA A1–1M is a version of
A1–1 that contains two changes at adjacent positions (GC to CG) in
the A1–1 sequence yielding an siRNA with two mismatches with
respect to the wild-type A1 mRNA. Ninety-six h after the first
transfection, total protein was isolated and the abundance of A1 and
A2 proteins was assessed by Western analysis using the anti-A1/A2
rabbit polyclonal antibody. When compared with control extracts, the
extracts from cells transfected with siRNAs A1–1, A1–2, A1–5, and
A1– 6 showed a marked reduction in the expression level of A1.
Similarly, except for A2– 4, all of the siRNAs against A2 promoted a
strong decrease in the A2 protein (Fig. 2A). As expected, the mismatched siRNA A1–1M did not affect the level of A1.
Next, we assessed whether the treatment with siRNAs affected cell
growth. Adherent and nonadherent cells derived from transfected and
control HeLa S3 cultures were counted 96 h after transfection (Fig.
2B). We also assessed cellular morphology by microscopic examination (Fig. 2C). Individual siRNAs that decreased either A1 or A2 did
not affect cell growth nor did they change cell morphology, even
when tested at a concentration of 300 nM (Fig. 2, B and C; data not
shown). Likewise, pairs of siRNAs that affected A1 or A2 alone did
not affect cell growth or cell morphology (e.g., A1– 6/A2– 4; data not
shown), and the A1–1M/A2–1 pair, which promotes a reduction in A2
but not A1, had no effect on growth and cell morphology (Fig. 2B).
However, combinations of siRNAs that promoted a reduction in the
abundance of both A1 and A2 (siRNAs A1–1/A2–1 and A1–5/A2–5;
Fig. 2B and data not shown, respectively) had a major impact on cell
counts, and cells displayed an altered morphology reminiscent of
apoptotic cells. In some experiments, the reduction in cell growth was
less apparent, but the majority of the cells were round and lost
adherence (data not shown). We attribute these variations between
experiments to differences in the timing of cell death. Trypan blue
exclusion staining indicated that the majority of the cells in the active
combinations of siRNAs always yielded a higher number of dead cells
(data not shown). For HeLa S3 cells, we typically used a concentration of 80 nM of siRNAs (40 nM of each siRNA when a mixture of two
was used). Although mixtures of siRNAs were also active in HeLa S3
cells at 20 nM, lower concentrations did not reduce cell viability.
Finally, we note that a 50% decrease in the intensity of both A1 and
A2 bands was almost invariably associated with massive cell death.
To confirm that cell death was occurring by apoptosis, we carried
out several indicative assays, including PARP and procaspase-3 cleavage assays, as well as DNA content analysis (Fig. 3). Only the
treatment with the pair of active siRNAs against A1 and A2 promoted
procaspase-3 and PARP cleavage (Fig. 3, A and B, respectively). We
also carried out a TUNEL assay, which specifically stains apoptotic
cells and which indicated that ⬎70% of the HeLa cells were TUNELpositive when treated with the A1–1/A2–1 siRNAs, as compared with
⬍0.1% when the treatment was performed with the control A1–1M/
A2–1 combination (Fig. 3C). The DNA content analysis showed the
characteristic sub-G1 increase because of DNA fragmentation with the
pair of A1–1/A2–1 siRNAs but not with the control A1–1M/A2–1
siRNAs (Fig. 3D). Thus, all of the tests support the conclusion that a
reduction in A1/A2 proteins in HeLa S3 cells promotes apoptosis.
The rapid cell death elicited by siRNAs targeting A1/A2 is consistent with the view that these proteins may function as telomeric
capping proteins. If this is the case, one of the first consequences
associated with a reduction in A1/A2 levels may be a shortening or
alteration of the single-stranded G-rich extension at telomeres. To
determine whether the siRNA treatment affected the single-stranded
extensions, we performed a T-OLA in HeLa S3 cells treated with
active and control siRNAs. When cells were mock-treated or treated
with a control pair of siRNAs (A1–1M/A2–1), the combined relative
percentage of ligation products corresponding to five or more oligos
is about the same as the percentage of ligation-products corresponding
to 3 and 4 oligos (Fig. 4, A and B). However, the distribution of
ligation products derived from cells treated with the active pair of
siRNAs (A1–1/A2–1) is quite distinct: the percentage of ligation
products with 5 or more oligos is now lower than the percentage
shorter products (see Fig. 4A for two independent experiments). A
similar result was observed when the analysis was performed 48-h
after transfection (data not shown). Importantly, the length distribution of the G-rich extensions did not change when HeLa S3 cells were
treated with staurosporine (Fig. 4B), an inducer of apoptosis.
7682
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
Fig. 2. siRNAs in HeLa S3 cells. Cells from the cervical carcinoma HeLa S3 cell line were seeded in six-well plates (65,000 cells/well) and were transfected twice at 24-h intervals.
Control samples were treated with oligofectamine in the absence of siRNA. A, Western analysis of A1/A2 expression. Cells were collected 96 h after the first transfection. Ponceau
S-staining of the nitrocellulose membrane was used to confirm equal protein loading (data not shown). The A1 and A2 proteins were revealed with the anti-A1/A2 antibody. B, 96 h
after transfection, both adherent and floating cells were harvested and counted. The gray area indicates that cells show an altered morphology characteristic of apoptosis. C, phase
contrast microscopy (⫻200 magnification) of HeLa S3 cells treated with siRNAs.
Impact of A1/A2-Targeted RNAi in Other Cancer Cell Lines.
The ability of RNAi to promote a reduction in A1 and A2 and to affect
cell viability was also investigated in cell lines derived from different
human cancers. We first tested the colorectal carcinoma cell line HCT
116 and a derivative that suffered the genetic ablation of the p53
alleles (HCT 116 p53⫺/⫺). siRNAs mixtures were applied to HCT
116 and HCT 116 p53⫺/⫺ cells as above. Cell viability was measured
72-h after transfection. Similar to what was observed for HeLa S3
cells, treatment with individual siRNAs promoted a reduction in the
targeted protein (Fig. 5A), but only the combination of siRNAs
targeting both A1 and A2 affected the growth and morphology of
HCT 116 cells (Fig. 5, B and C). The mismatched A1–1M/A2–1
siRNAs promoted a reduction in A2 only and did not affect cell counts
or cell morphology. The apoptotic phenotype obtained with the pair of
siRNAs that affected both A1 and A2 was confirmed by testing for
PARP and procaspase-3 cleavage (data not shown). Notably, the
p53⫺/⫺ version of HCT 116 was equally sensitive to abrogation of
A1/A2 expression (Fig. 5B). The analysis of DNA content revealed an
increased number of cells in the sub-G1 population, consistent with
apoptosis-mediated chromatin fragmentation (Fig. 5D). HCT 116
cells grown at low concentrations of serum were as sensitive to siRNA
against A1/A2 (data not shown). The fibrosarcoma cell line HT-1080
was also tested. The siRNA-mediated reduction in A1/A2 expression
correlated with a reduction in cell growth and a change in cell
morphology characteristic of apoptosis (Fig. 5, B and C).
Additional cancer cell lines that were tested include an ovarian
carcinoma cell line (PA-1), a metastatic ovarian carcinoma cell line
(SK-OV-3), a glioblastoma cell line (U-373 MG), and a breast carcinoma cell line (MCF-7; Fig. 6). Notably, the SK-OV-3 cell line is p53
null, whereas U-373 MG cells express a mutated form of p53 (40, 43).
In all of the cases, only the treatment with the pair of siRNAs
A1–1/A2–1 elicited a marked reduction in the expression of A1 and
A2, and this reduction was accompanied by a reduction in cell growth
and a phenotypic change characteristic of apoptosis (Fig. 6; data not
shown).
The RNAi-Mediated Reduction in A1/A2 Does Not Affect the
Growth of Nontransformed Human Cell Lines. To evaluate the
impact of a reduction in A1/A2 levels in normal mortal cells, we
used three cell lines: foreskin fibroblasts (BJ cells), colonic CCD18Co myofibroblasts, and the epithelial intestinal cell line HIEC.
We also used the BJ-TIELF cell line, which is an immortalized
derivative of the BJ-line that expresses the catalytic component
(human telomerase reverse transcriptase) of the human telomerase
(44, 45). All of these cells express hnRNP A1/A2 proteins (Fig.
7A). We noted that the immortal BJ-TIELF cell line expresses
more A1/A2 than earlier passages of BJ cells (data not shown),
consistent with the view that A1/A2 expression drops as mortal
cells approach senescence (46). RNAi assays with siRNAs targeting A1, A2, or both promoted a reduction in the corresponding
proteins that was comparable with the drop obtained with cancer
cell lines (Fig. 7A). In contrast to cancer cells, however, all of the
mortal cell lines tolerated well a reduction in A1/A2, and the
7683
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
treatment had little effect on cell growth and morphology (Fig. 7,
B and C). Even the growth of the immortal but nontransformed
BJ-TIELF cells was not affected by a strong reduction in A1/A2
(Fig. 7A). In all cases, cell cycle analysis of the DNA content
indicated no significant increases in sub-G1 DNA content (Fig. 7D;
data not shown). However, in experiments using BJ-TIELF cells,
we occasionally noted a slight increase in the G2-M population
(Fig. 7D). We also performed the T-OLA assay on DNA derived
from CCD-18Co cells that were mock treated, and treated with
Fig. 3. Apoptosis in HeLa S3 cells. A, HeLa S3 cells were transfected as described
earlier, and cells were harvested 96 h after the first transfection. The Western blot was
probed with a mixture of monoclonal antibodies that recognize both the Mr 33,000
inactive procaspase-3 and the activated Mr 20,000 form found in apoptotic cells. B, the
same Western analysis as shown in A but probed with an antibody that recognizes the
PARP enzyme, which is a substrate for the activated caspase-3 (52, 53). C, HeLa S3 cells
were transfected with the indicated siRNAs or treated with 1 ␮M staurosporine. Ninety-six
h after the first transfection or 15 h after staurosporine treatment, TUNEL labeling was
performed. Propidium iodide was used as a nuclear counterstain to visualize the whole cell
population. Apoptotic nuclei were labeled with fluorescein and appeared green, whereas
living cells were stained red. Note that when cells are treated with both siA1 and siA2,
there is a major decrease in the cell number as observed previously and that most of the
remaining cells are positive for TUNEL labeling. D, siRNA-treated cells were fixed and
stained with propidium iodide before DNA content analysis by cytometry. n refers to the
haploid DNA content. Note the appearance of sub-G1 DNA, associated with apoptosis, in
HeLa S3 cells. siA1, A1–1; siA2, A1–2; siA1M, A1–1M.
Fig. 4. T-OLA assay to measure the distribution of lengths of telomeric G-tails. A, 72 h
after the first transfection, HeLa S3 cells were harvested and cellular DNA was extracted,
and a T-OLA assay was performed. The results of two independent experiments are
shown, the gel on the left depicting the results obtained in experiment 2. B, HeLa S3 cells
were treated with staurosporine/DMSO or DMSO alone for 28 h before performing the
T-OLA assay. S, staurosporine-treated cells; D, DMSO-treated cells. All oligo ligation
assays were performed using 10 ␮g of cellular DNA and ligation products were resolved
on a sequencing gel, detected by autoradiography and quantitated as described in “Materials and Methods.” The numbers on the right of the gels indicate the number of oligos
in some ligation products. Values for ligation products representing the ligation of 3 and
4 oligos or 5–13 oligos were pooled and plotted as frequency (in percentages). siA1, A1–1;
siA2, A1–2; siA1M, A1–1M.
7684
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
Fig. 5. Impact of the treatment with siRNAs on HCT 116, HCT 116 p53⫺/⫺, and HT-1080 cancer cell lines. A, 72 h after transfection, cells were harvested and processed to
determine the impact of RNAi on A1 and A2 expression. Western blot analysis was performed with the polyclonal anti-A1/A2 antibody. B, effect of the siRNA treatments on cell growth
as measured by population doublings. The gray area indicates that cells displayed an altered morphology reminiscent of apoptotic cells. C, phase contrast microscopy (magnification,
⫻200) of cells treated with siRNAs. D, the DNA content profile of HCT 116 p53⫺/⫺ cells treated with siRNAs is shown.
control and active pairs of siRNAs (A1–1M/A2–1 and A1–1/A2–1,
respectively; Fig. 7E). Consistent with the results showing no cell
mortality on treatment with the active pair of siRNAs, there was no
discernible difference in the T-OLA products between the three
treatments. We conclude that in contrast to cancer cell lines, mortal
human cell lines tolerate well the reduction in A1/A2 proteins
imposed by RNAi.
DISCUSSION
The successful treatment of cancer rests on the identification of
targets that are specifically expressed in cancer cells and also play an
important role in promoting or allowing unlimited cell division. Although interesting targets with such properties have been identified in
cancer cells of different origins, there are few examples of factors that
play important roles in many types of cancers. Here, we present
evidence that the hnRNP A1/A2 proteins could represent potential
new and broad targets against cancer.
First, we report that the expression of A1/A2 is elevated in the
nuclei of cells derived from a variety of cancers (Fig. 1; Table 1).
Moderate to high levels of hnRNP A1 proteins were detected in
breast, prostate, ovary, pancreas, and skin cancer biopsies. These data
confirm and extend previous studies that have documented the ex-
pression of A1 and A2/B1 proteins in colon and lung cancers, respectively (33, 35–38). Most significantly, however, we show that the
level of A1 and A2 is comparatively lower, and in many instances
virtually absent, in normal somatic tissues, although A1/A2 expression can be detected in some specific cell types (Fig. 1; Table 2). Only
the basal layer of the normal skin displayed high level expression of
hnRNP A1/A2. However, it is likely that A1/A2 will be similarly
expressed in other highly proliferative cell types.
Second, the preferential expression of A1/A2 in cancer cells led us
to investigate the impact of a reduction in A1/A2 in different human
cancer cell lines. Using siRNAs to promote specific decreases in
A1/A2, we observed that such treatments elicited programmed cell
death of HeLa cells. Remarkably, cancer cell lines derived from colon,
ovarian, breast, and brain cancers were all similarly sensitive to a
reduction in A1/A2 protein levels. Notably, a reduction in A1 alone or
in A2 alone did not promote cell death. The importance of a combined
reduction in both A1 and A2 suggests that A1 and A2 are functional
homologues. Therefore, hnRNP A2 compensation would explain the
survival of the mouse erythroleukemic CB3 cell line, which is severely deficient in A1 (7). Thus, in a situation where A1 and A2 are
expressed in similar amounts, reducing the global level of A1/A2 by
targeting either A1 or A2 does not affect cell growth, and only the
7685
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
Fig. 6. Impact of the siRNA treatments on PA-1, U-373 MG, SK-OV-3, and MCF-7 cells. A, 96 h after transfection, cells were harvested and processed to determine the impact
of RNAi on A1 and A2 expression. Western blot analysis was performed with the polyclonal anti-A1/A2 antibody. B, effect of the siRNA treatments on cell growth as measured by
population doublings. The gray area indicates that cells displayed a drastic change in morphology. C, phase contrast microscopy (magnification, ⫻200 except for SK-OV-3, ⫻100)
of cells treated with siRNAs. n.d., not determined.
dual targeting of A1 and A2 allows a substantial decrease in the
overall levels of A1/A2.
The rapidity with which cancer cells die after the treatment with
siRNAs against A1 and A2 is consistent with the view that the A1/A2
proteins play a direct role as telomeric capping factors. In support of
this view, we have shown that abrogating A1/A2 expression is accompanied by a change in the length distribution of telomeric singlestranded G-rich extensions (G-tails). Because this change can be
detected 48 –72 h after the first application of siRNAs, we hypothesize
that it is a crucial event to trigger apoptosis in cells that rely on A1/A2
as capping factors. Consistent with this view, normal cells that support
well a significant reduction in A1/A2 do not show such a change in
the size distribution of T-OLA products, suggesting that the A1/A2
proteins may be less important for capping of the G-tails in such cells.
Notably, such changes in the integrity of G-tails are not observed
when cells are treated with the apoptotic inducer staurosporine, suggesting that the degradation of G-tails is not an obligatory feature or
consequence associated with apoptosis, and reinforcing the conclusion
that the drop in A1/A2 is directly responsible for the changes at the
G-tails. Although we favor the proposition that shortening of G-tails
is an important step to initiate the apoptotic response, it remains
possible that alterations in other A1/A2-mediated processes contribute
or even cause apoptosis.
The observed induction of apoptosis in cancer cells appears to be
independent of the status of p53 expression, because p53 null and p53
mutant cell lines are also very sensitive to RNAi against A1/A2.
Notably, apoptosis triggered by a dominant-negative version of the
telomeric factor TRF2 required a wild-type p53 protein (47). Therefore, the outcome of a drop in A1/A2 is more similar to the impact of
telomerase inhibition on the growth of human cancer cells, which also
is independent of the status of p53 (19).
In contrast to the situation in cancer cells, the siRNA-mediated
reduction in A1/A2 levels in normal and mortal cell lines did not
affect cell division and did not provoke cell death. The fact that these
“normal” cell lines are more resistant to a loss of A1/A2 function is
intriguing, and may indicate that the capping complex at telomeres is
different between cancer and normal cells. This would not necessarily
be unexpected, because the enzyme telomerase is expressed at appreciable levels in cancer, but not in normal cells. It has been shown
recently that this enzyme localizes to the telomeres where it may play
7686
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
Fig. 7. siRNA targeting of A1/A2 in normal cell
lines. BJ, CCD-18Co, and HIEC are normal mortal
cell lines, whereas the BJ-TIELF cell line is immortal because it expresses the catalytic subunit of
telomerase. Cells were seeded in a six-well plate
and were transfected twice with the indicated
siRNA alone (80 nM) or with combinations of
siRNA (40 nM each siRNA for a total concentration
of 80 nM). Control cells were treated with oligofectamine in the absence of siRNA. A, Western
analysis with the polyclonal antibody against A1/
A2. Ponceau S-staining of the nitrocellulose membrane was used to confirm equal protein loading
(data not shown). B, Ninety-six h after transfection,
both adherent and floating cells were harvested and
counted. C, phase contrast microscopy of adherent
cells (magnification, ⫻200). D, DNA content analysis of BJ-TIELF and HIEC cells treated with
siRNA against A1/A2, a treatment with A1–1M/
A2–1 and oligofectamine alone (control). The 96-h
post-transfection profiles are compared with a parallel treatment of HeLa S3 cells. E, T-OLA assay to
measure the distribution of lengths of telomeric
G-tails. Ninety-six h after the first transfection with
the indicated pairs of siRNAs (160 nM), the DNA
of CCD-18Co cells was used to perform a T-OLA
assay. Ligation products were resolved on a gel and
quantitated. Values for ligation products representing the ligation of 3 and 4 oligos or ⱖ5 oligos are
expressed in frequency (percentages). siA1, A1–1;
siA2, A1–2; siA1M, A1–1M.
a role in the capping function (48 –50). Therefore, telomerase expression and other mechanisms may lead to differences in the size of the
single-stranded extensions, which may affect the identity and function
of capping factors. We speculate that such differences include the
A1/A2 proteins, because their abrogation by siRNA affects the length
distribution of the G-tails. A similar decrease in the size of G-tails has
been associated recently with replicative senescence of normal human
cells in culture (12), and providing oligos complementary to G-tails to
cells in culture induces damage-response pathways (51). These data
point to a critical importance of the integrity of the G-tails for the
capping function of telomeres. Therefore, our results are consistent
with the hypothesis that abrogating the expression or inhibiting the
function of cancer cell-specific G-tail binding proteins, such as A1/
A2, could entail a severe reduction in the capping function and explain
the cellular phenotypes observed in the present study.
In summary, our results demonstrate that a combined reduction of
hnRNP A1/A2 causes programmed cell death in a variety of cancer
cell lines, including p53-compromised cells. Our findings establish
A1/A2 as potential drug targets in cancer therapeutics and provide a
strong rationale for the development of strategies aimed at abrogating
the expression or activity of hnRNP A1/A2 proteins in cancer cells.
Such approaches should be particularly attractive, given that A1/A2
are expressed at low levels in normal tissues and that normal mortal
cells can tolerate a reduction in A1/A2 levels without significant
effects on cell viability.
ACKNOWLEDGMENTS
We thank D. Fortin, C. Rancourt, B. Vogelstein, and K. W. Kinzler for cell
lines. We thank Silvia Bacchetti and Sam Benchimol for comments on the
manuscript.
REFERENCES
1. Chakhparonian, M., and Wellinger, R. J. Telomere maintenance and DNA replication:
how closely are these two connected? Trends Genet., 19: 439 – 446, 2003.
2. McElligott, R., and Wellinger, R. J. The terminal DNA structure of mammalian
chromosomes. EMBO J., 16: 3705–3714, 1997.
3. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., and
de Lange, T. Mammalian telomeres end in a large duplex loop. Cell, 97: 503–514,
1999.
4. van Steensel, B., and de Lange, T. Control of telomere length by the human telomeric
protein TRF1. Nature (Lond.), 385: 740 –743, 1997.
7687
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
hnRNP A1/A2 PROTEINS AS TARGETS AGAINST CANCER
5. van Steensel, B., Smogorzewska, A., and de Lange, T. TRF2 protects human telomeres from end-to-end fusions. Cell, 92: 401– 413, 1998.
6. Ishikawa, F., Matunis, M. J., Dreyfuss, G., and Cech, T. R. Nuclear proteins that bind
the pre-mRNA 3⬘ splice site sequence r(UUAG/G) and the human telomeric DNA
sequence d(TTAGGG)n. Mol. Cell. Biol., 13: 4301– 4310, 1993.
7. LaBranche, H., Dupuis, S., Ben-David, Y., Bani, M. R., Wellinger, R. J., and Chabot,
B. Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric
repeats and telomerase. Nat. Genet., 19: 199 –202, 1998.
8. McKay, S. J., and Cooke, H. hnRNP A2/B1 binds specifically to single stranded
vertebrate telomeric repeat TTAGGGn. Nucleic Acids Res, 20: 6461– 6464, 1992.
9. Baumann, P., Podell, E., and Cech, T. R. Human Pot1 (protection of telomeres)
protein: cytolocalization, gene structure, and alternative splicing. Mol. Cell. Biol., 22:
8079 – 8087, 2002.
10. Baumann, P., and Cech, T. R. Pot1, the putative telomere end-binding protein in
fission yeast and humans. Science (Wash. DC), 292: 1171–1175, 2001.
11. Karlseder, J., Smogorzewska, A., and de Lange, T. Senescence induced by altered
telomere state, not telomere loss. Science (Wash. DC), 295: 2446 –2449, 2002.
12. Stewart, S. A., Ben-Porath, I., Carey, V. J., O’Connor, B. F., Hahn, W. C., and
Weinberg, R. A. Erosion of the telomeric single-strand overhang at replicative
senescence. Nat. Genet., 33: 492– 496, 2003.
13. Masutomi, K., Yu, E. Y., Khurts, S., Ben-Porath, I., Currier, J. L., Metz, G. B.,
Brooks, M. W., Kaneko, S., Murakami, S., DeCaprio, J. A., Weinberg, R. A., Stewart,
S. A., and Hahn, W. C. Telomerase maintains telomere structure in normal human
cells. Cell, 114: 241–253, 2003.
14. de Lange, T., and Jacks, T. For better or worse? Telomerase inhibition and cancer.
Cell, 98: 273–275, 1999.
15. Harley, C. B., Kim, N. W., Prowse, K. R., Weinrich, S. L., Hirsch, K. S., West, M. D.,
Bacchetti, S., Hirte, H. W., Counter, C. M., Greider, C. W., et al. Telomerase, cell
immortality, and cancer. Cold Spring Harb. Symp. Quant. Biol., 59: 307–315, 1994.
16. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L.,
Coviello, G. M., Wright, W. E., Weinrich, S. L., and Shay, J. W. Specific association
of human telomerase activity with immortal cells and cancer. Science (Wash. DC),
266: 2011–2015, 1994.
17. Reddel, R. R., Bryan, T. M., Colgin, L. M., Perrem, K. T., and Yeager, T. R.
Alternative lengthening of telomeres in human cells. Radiat. Res., 155: 194 –200,
2001.
18. Reddel, R. R., Bryan, T. M., and Murnane, J. P. Immortalized cells with no detectable
telomerase activity. A review. Biochemistry (Mosc.), 62: 1254 –1262, 1997.
19. Hahn, W. C., Stewart, S. A., Brooks, M. W., York, S. G., Eaton, E., Kurachi, A.,
Beijersbergen, R. L., Knoll, J. H., Meyerson, M., and Weinberg, R. A. Inhibition of
telomerase limits the growth of human cancer cells. Nat. Med., 5: 1164 –1170, 1999.
20. Herbert, B., Pitts, A. E., Baker, S. I., Hamilton, S. E., Wright, W. E., Shay, J. W., and
Corey, D. R. Inhibition of human telomerase in immortal human cells leads to
progressive telomere shortening and cell death. Proc. Natl. Acad. Sci. USA, 96:
14276 –14281, 1999.
21. Feng, J., Funk, W. D., Wang, S. S., Weinrich, S. L., Avilion, A. A., Chiu, C. P.,
Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., et al. The RNA component of human
telomerase. Science (Wash. DC), 269: 1236 –1241, 1995.
22. Pruzan, R., Pongracz, K., Gietzen, K., Wallweber, G., and Gryaznov, S. Allosteric
inhibitors of telomerase: oligonucleotide N3⬘–⬎P5⬘ phosphoramidates. Nucleic Acids Res., 30: 559 –568, 2002.
23. Gryaznov, S., Pongracz, K., Matray, T., Schultz, R., Pruzan, R., Aimi, J., Chin, A.,
Harley, C., Shea-Herbert, B., Shay, J., Oshima, Y., Asai, A., and Yamashita, Y.
Telomerase inhibitors– oligonucleotide phosphoramidates as potential therapeutic
agents. Nucleosides Nucleotides Nucleic Acids, 20: 401– 410, 2001.
24. Lichtsteiner, S. P., Lebkowski, J. S., and Vasserot, A. P. Telomerase. A target for
anticancer therapy. Ann. N. Y. Acad. Sci., 886: 1–11, 1999.
25. Pascolo, E., Wenz, C., Lingner, J., Hauel, N., Priepke, H., Kauffmann, I., GarinChesa, P., Rettig, W. J., Damm, K., and Schnapp, A. Mechanism of human telomerase
inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate. J. Biol. Chem.,
277: 15566 –15572, 2002.
26. Damm, K., Hemmann, U., Garin-Chesa, P., Hauel, N., Kauffmann, I., Priepke, H.,
Niestroj, C., Daiber, C., Enenkel, B., Guilliard, B., Lauritsch, I., Muller, E., Pascolo,
E., Sauter, G., Pantic, M., Martens, U. M., Wenz, C., Lingner, J., Kraut, N., Rettig,
W. J., and Schnapp, A. A highly selective telomerase inhibitor limiting human cancer
cell proliferation. EMBO J., 20: 6958 – 6968, 2001.
27. de Lange, T. Protection of mammalian telomeres. Oncogene, 21: 532–540, 2002.
28. Blackburn, E. H. Telomere states and cell fates. Nature (Lond.), 408: 53–56, 2000.
29. Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. hnRNP proteins and
the biogenesis of mRNA. Annu. Rev. Biochem., 62: 289 –321, 1993.
30. Dallaire, F., Dupuis, S., Fiset, S., and Chabot, B. Heterogeneous nuclear ribonucleoprotein A1 and UP1 protect mammalian telomeric repeats and modulate telomere
replication in vitro. J. Biol. Chem., 275: 14509 –14516, 2000.
31. Fiset, S., and Chabot, B. hnRNP A1 may interact simultaneously with telomeric DNA
and the human telomerase RNA in vitro. Nucleic Acids. Res., 29: 2268 –2275, 2001.
32. Biamonti, G., Bassi, M. T., Cartegni, L., Mechta, F., Buvoli, M., Cobianchi, F., and
Riva, S. Human hnRNP protein A1 gene expression. Structural and functional
characterization of the promoter. J. Mol. Biol., 230: 77– 89, 1993.
33. Zhang, L., Zhou, W., Velculescu, V. E., Kern, S. E., Hruban, R. H., Hamilton, S. R.,
Vogelstein, B., and Kinzler, K. W. Gene expression profiles in normal and cancer
cells. Science (Wash. DC), 276: 1268 –1272, 1997.
34. Iervolino, A., Santilli, G., Trotta, R., Guerzoni, C., Cesi, V., Bergamaschi, A.,
Gambacorti-Passerini, C., Calabretta, B., and Perrotti, D. hnRNP A1 nucleocytoplasmic shuttling activity is required for normal myelopoiesis and BCR/ABL leukemogenesis. Mol. Cell. Biol., 22: 2255–2266, 2002.
35. Zhou, J., Mulshine, J. L., Unsworth, E. J., Scott, F. M., Avis, I. M., Vos, M. D., and
Treston, A. M. Purification and characterization of a protein that permits early
detection of lung cancer. Identification of heterogeneous nuclear ribonucleoproteinA2/B1 as the antigen for monoclonal antibody 703D4. J. Biol. Chem., 271: 10760 –
10766, 1996.
36. Satoh, H., Kamma, H., Ishikawa, H., Horiguchi, H., Fujiwara, M., Yamashita, Y. T.,
Ohtsuka, M., and Sekizawa, K. Expression of hnRNP A2/B1 proteins in human
cancer cell lines. Int. J. Oncol., 16: 763–767, 2000.
37. Sueoka, E., Goto, Y., Sueoka, N., Kai, Y., Kozu, T., and Fujiki, H. Heterogeneous
nuclear ribonucleoprotein B1 as a new marker of early detection for human lung
cancers. Cancer Res., 59: 1404 –1407, 1999.
38. Snead, D., Perunovic, B., Cullen, N., Needham, M., Dhillon, D., Satoh, H., and
Kamma, H. hnRNP B1 expression in benign and malignant lung disease. J. Pathol.,
200: 88 –94, 2003.
39. Yan-Sanders, Y., Hammons, G. J., and Lyn-Cook, B. D. Increased expression of
heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) in pancreatic tissue from
smokers and pancreatic tumor cells. Cancer Lett., 183: 215–220, 2002.
40. Yaginuma, Y., and Westphal, H. Abnormal structure and expression of the p53 gene
in human ovarian carcinoma cell lines. Cancer Res., 52: 4196 – 4199, 1992.
41. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T.
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian
cells. Nature (Lond.), 411: 494 – 498, 2001.
42. Cimino-Reale, G., Pascale, E., Battiloro, E., Starace, G., Verna, R., and D’Ambrosio,
E. The length of telomeric G-rich strand 3⬘-overhang measured by oligonucleotide
ligation assay. Nucleic Acids Res., 29: E35, 2001.
43. Rothmann, T., Hengstermann, A., Whitaker, N. J., Scheffner, M., and zur Hausen, H.
Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53
status in tumor cells. J. Virol., 72: 9470 –9478, 1998.
44. Vaziri, H., Squire, J. A., Pandita, T. K., Bradley, G., Kuba, R. M., Zhang, H., Gulyas,
S., Hill, R. P., Nolan, G. P., and Benchimol, S. Analysis of genomic integrity and
p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts. Mol. Cell. Biol., 19: 2373–2379, 1999.
45. Vaziri, H., and Benchimol, S. Reconstitution of telomerase activity in normal human
cells leads to elongation of telomeres and extended replicative life span. Curr. Biol.,
8: 279 –282, 1998.
46. Hubbard, K., Dhanaraj, S. N., Sethi, K. A., Rhodes, J., Wilusz, J., Small, M. B., and
Ozer, H. L. Alteration of DNA and RNA binding activity of human telomere binding
proteins occurs during cellular senescence. Exp. Cell Res., 218: 241–247, 1995.
47. Karlseder, J., Broccoli, D., Dai, Y., Hardy, S., and de Lange, T. p53- and ATMdependent apoptosis induced by telomeres lacking TRF2. Science (Wash. DC), 283:
1321–1325, 1999.
48. Yang, J., Chang, E., Cherry, A. M., Bangs, C. D., Oei, Y., Bodnar, A., Bronstein, A.,
Chiu, C. P., and Herron, G. S. Human endothelial cell life extension by telomerase
expression. J. Biol. Chem., 274: 26141–26148, 1999.
49. Sharma, G. G., Gupta, A., Wang, H., Scherthan, H., Dhar, S., Gandhi, V., Iliakis, G.,
Shay, J. W., Young, C. S., and Pandita, T. K. hTERT associates with human
telomeres and enhances genomic stability and DNA repair. Oncogene, 22: 131–146,
2003.
50. Zhu, J., Wang, H., Bishop, J. M., and Blackburn, E. H. Telomerase extends the
lifespan of virus-transformed human cells without net telomere lengthening. Proc.
Natl. Acad. Sci. USA, 96: 3723–3728, 1999.
51. Li, G. Z., Eller, M. S., Firoozabadi, R., and Gilchrest, B. A. Evidence that exposure
of the telomere 3⬘ overhang sequence induces senescence. Proc. Natl. Acad. Sci.
USA, 100: 527–531, 2003.
52. Tewari, M., Quan, L. T., O’Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R.,
Poirier, G. G., Salvesen, G. S., and Dixit, V. M. Yama/CPP32 ␤, a mammalian
homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate
poly(ADP-ribose) polymerase. Cell, 81: 801– 809, 1995.
53. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant,
M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., et al. Identification and
inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature
(Lond.), 376: 37– 43, 1995.
7688
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.
Small Interfering RNA-Mediated Reduction in Heterogeneous
Nuclear Ribonucleoparticule A1/A2 Proteins Induces
Apoptosis in Human Cancer Cells but not in Normal Mortal
Cell Lines
Caroline Patry, Louise Bouchard, Pascale Labrecque, et al.
Cancer Res 2003;63:7679-7688.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/63/22/7679
This article cites 51 articles, 23 of which you can access for free at:
http://cancerres.aacrjournals.org/content/63/22/7679.full#ref-list-1
This article has been cited by 29 HighWire-hosted articles. Access the articles at:
http://cancerres.aacrjournals.org/content/63/22/7679.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 2003 American Association for Cancer
Research.